Method, monoclonal antibody, and monoclonal antibody fragments for detecting human non-small cell lung carcinomas and cell line for producing such antibodies

ABSTRACT

The present invention is concerned with two novel monoclonal antibodies which define carbohydrate antigens associated with human non-small cell lung carcinomas (&#34;NSCLC&#34;) and certain other human carcinomas. The antibodies bind to normal human cells to a much lesser degree than to tumor cells. The antibodies find use in diagnostic methods such as the detection of malignant cells associated with NSCLC and in therapeutic methods. The invention also comprises a method for determining the presence of a malignant condition in the lung of a subject. The method involves examining tissue from the subject for the presence of antigens which are Le x  or Le y  antigen or which have the characteristics of Le y  and Le x .

BACKGROUND OF THE INVENTION

1. Field of the Invention

Human lung carcinomas are responsible for most deaths from cancer amongmen and are in the process of overtaking breast carcinomas as the mostfrequent cause of cancer death among women (Cancer Facts and Figures,1983). This disease can be divided into 4 major histological types,i.e., epidermoid (30%), adenocarcinoma (35%), large-cellundifferentiated (15%), and small-cell (20%). Most cases of lungcarcinomas are incurable by chemotherapy and radiation therapy. Smallcell lung carcinomas may respond to chemotherapy and radiation therapyby a reduction in size, but not a total cure. Complete surgical removalof the tumor appears to be the only effective therapy. Unfortunately,however, fewer than 30% of lung cancer patients have tumors which can betotally resected at diagnosis and of these, fewer than one-third survive5 years after apparent complete surgical removal of all tumor. There isa great need, therefore, for methods that would make possible an earlierdiagnosis of lung cancer, a better definition of the degree of cancerspread, and a more effective therapy.

Monoclonal antibodies may be used for all these purposes. Aprerequisite, however, is to find antibodies to antigens that are morestrongly expressed in lung cancer than in normal adult tissues. In viewof the known heterogeneity of tumor cell populations, the presence ofseveral determinants on the same antigen molecule, the anticipateddifferences between antigens with respect to their suitability asdiagnostic markers as compared to therapeutic targets, and the differentbiological characteristics of different antibodies to the same antigen,a number of different antibodies may be needed.

2. Description of the Prior Art

Human monoclonal antibodies to lung cancer antigens are described bySikora et al., Br. J. Cancer (1981) 43: 696-700. Monoclonal antibodiesthat demonstrate specificity for several types of human lung cancer aredisclosed by Cuttitta et al., Proc. Natl. Acad. Sci. U.S.A. (1981) 78:4591-4595. Antigens associated with a human lung adenocarcinoma definedby monoclonal antibodies are described by Varki et al., Cancer Research(1984) 44: 681-687.

Mouse monoclonal antibodies to glycolipid Le^(x) antigens are describedby Hakomori et al., in Biochemical and Biophysical ResearchCommunications (1981) 100: 1578-1586; Brockhaus et al., Arch. Biochem.Biophys. (1982) 217: 647; Fukushi et al., J. Biol. Chem. (1984) 259:506; Fukushi et al. J. Exp. Med. (1984) 159: 506; Chia et al., CancerRes. (1985) 45: 435. Antibodies to Le^(y) antigens have been describedby Abe et al., J. Biol. Chem (1983) 258: 8934; Lloyd et al.,Immunogenetics (1983) 17: 537; Brown et al., Biosci. Rep. (1983) 3: 163.

Continuous cultures of fused cells secreting antibody of predefinedspecificity are described by Kohler et al., Nature (1975) 265: 495-497.

SUMMARY OF THE INVENTION

The present invention is concerned with novel monoclonal antibodieswhich define determinant sites on carbohydrate (glycolipid) antigensassociated with human non-small cell lung carcinoma (NSCLC) cells. Theterm "NSCLC cells" includes epidermoid carcinoma cells, adenocarcinomacells, and large cell undifferentiated carcinoma cells. The determinantsite may also be found on antigens of some other carcinomas, e.g., somecarcinomas of the breast, and, thus, the antibodies of the inventionwill also bind to these other carcinoma cells. The present monoclonalantibodies bind to a much lesser degree to normal adult cells than totumor cells. The term "bind to a much lesser degree" means that thebinding will not be detectable by immunohistological techniques and thatthere will be much weaker binding to normal cells than to tumor cells.The monoclonal antibodies are secreted by murine hybridomas.

The present invention also includes methods for determining the presenceof a malignant condition in the lung by examining lung tissue and otherhuman tissue for the presence of an antigen having the characteristicsof Le^(x) or Le^(y) antigens. The Le^(x) antigen has been described byHakomori et al., supra; Brockhaus et al., supra; Fukushi et al., supra;and Chia et al., supra; The Le^(y) antigen has been described by Abe etal., supra, Lloyd et al., supra, and Brown et al., supra.

For example, tissue from a subject can be contacted with an antibodywhich defines a determinant site on a cell associated carbohydrateantigen which is Le^(y) or Le^(x) or which has the characteristics ofLe^(y) or Le^(x) or a functional equivalent or fragment of suchantibody.

Thus, the invention concerns certain diagnostic methods employing themonoclonal antibodies of the invention. One such method involves thedetermination of the presence of NSCLC cells in a specimen suspected ofcontaining such cells. The specimen is contacted with the monoclonalantibody, which is capable of distinguishing such cells from other celltypes which may be present in the specimen. The contact is carried outunder conditions for binding of the antibody to such cells. Aftercontact, the presence or absence of binding of the antibody to the cellsin the specimen is determined. This binding is related to the presenceor absence of the NSCLC cells in the specimen. Generally, the specimenis contacted with a labeled specific binding partner for the monoclonalantibody. This label is capable of producing a detectable signal.Another diagnostic method involves the localization to a tumor ofantibodies or antibody fragments which have been properly labelled (e.g.with a radioisotope) and are subsequently injected into patients. Thismethod can provide better ways to stage cancer patients with respect toextent of disease and to monitor changes in response to therapy.

The invention also has therapeutic applications. The antibodies canreact with either or both of the above-mentioned antigens that areexpressed in high concentrations at the tumor cell surface. It can beused as a carrier of various agents which have an anti-tumor effect,including, but not restricted to, chemotherapeutic drugs andradioisotopes.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention concerns novel antibodies which bind to an antigenon human NSCLC cells and certain diagnostic and therapeutic methodsemploying these antibodies. The monoclonal antibodies of the inventionmay be produced according to the standard techniques of Kohler andMilstein, supra. For example, human lung carcinoma cells from pluraleffusions or cultured cells from human non-small cell lung carcinoma, orcells from a normal fetal lung, are used as the immunogen. These cellsare injected into a mouse and, after a sufficient time, the mouse issacrificed and spleen cells obtained. The spleen cell chromosomesencoding desired immunoglobulins are immortalized by fusing the spleencells with myeloma cells or with lymphoma cells, generally in thepresence of polyethylene glycol. The resulting cells, which include thefused hybridomas, are allowed to grow in a selective medium, such asHAT-medium, and the surviving cells are grown in such medium usinglimiting dilution conditions. The cells are grown in a suitablecontainer, e.g., microtiter wells, and the supernatant is screened formonoclonal antibodies having the desired specificity.

Various techniques exist for enhancing yields of monoclonal antibodies,such as injection of the hybridoma cells into a peritoneal cavity of amammalian host, which accepts the cells, and harvesting the ascitesfluid. Where an insufficient amount of the monoclonal antibody collectsin the ascites fluid, the antibody is harvested from the blood of thehost. Various conventional ways exist for isolation and purification ofthe monoclonal antibodies, so as to free the monoclonal antibodies fromother proteins and other contaminants (see Kohler and Milstein, supra).

One monoclonal antibody in accordance with the present invention isdesignated L15. It defines a cell surface carbohydrate antigen which is,or which has the characteristics of, Le^(y) antigen. We have identifiedthis antigen as characteristic of human NSCLC cells and cells fromcertain other human carcinomas. The antibody is of the IgM isotype. Itdoes not bind detectably to normal cells, such as fibroblasts,endothelial cells, or epithelial cells in the major organs. The L15antibody is produced by the L15 murine hybridoma.

Another monoclonal antibody in accordance with the present invention isdesignated L17. It defines a cell surface carbohydrate determinant whichis, or which has the characteristics of, Le^(x) antigen. We haveidentified this antigen also as characteristic of human NSCLC cells andcells from certain other human carcinomas. This antibody is of the IgMisotype. It binds less to normal cells, such as fibroblasts, endothelialcells, or epithelial cells in the major organs than to tumor cells. TheL17 antibody is produced by the L17 murine hybridoma.

Antibody L15 recognizes antigen Le^(y) which has the structure asidentified below: ##STR1## A series of glycolipids with different chainlengths can be detected by this antibody.

Antibody L17 reacts with a series of glycolipid antigens, all of whichcarry Le^(x) at the terminus, i.e.: ##STR2## R can be various lengths ofblood group type 2 chain

    (Galβ1→4GlcANcβ1→3Galβ1→4GlcNacβ1.fwdarw.3).

The antibody also reacts with trifucosyl Y structure as shown below:##STR3##

Also included within the scope of the invention are useful bindingfragments of the above monoclonal antibodies such as Fab, F(ab')₂, Fvfragments and so forth. The antibody fragments are obtained byconventional techniques. For example, useful binding fragments may beprepared by peptidase digestion of the antibody using papain or pepsin.

The invention also includes the diagnostic and therapeutic use, indetecting and treating NSCLC carcinoma in humans, of Le^(y) or Le^(x)antigen or antigens having the characteristics of Le^(x) and Le^(y)antigens, that is, antigens which are related but not identical toLe^(y) or Le^(x) and which are recognized by an antibody that alsorecognizes Le^(y) or Le^(x). The antigen can be purified by conventionalmethods such as described by Hakomori et al., supra, and by Abe et al.,supra.

One method of the invention involved the determination of the presenceof a malignant condition in the lung of a subject by examining lungtissue or other tissue from the subject for the presence of acarbohydrate antigen, which is Le^(y) or Le^(x) or which has thecharacteristics of Le^(y) or Le^(x). The term "tissue" refers to excisedcells, exfoliative cells, body fluids such as blood, plasma, serum,urine, etc., and the like. The term "malignant condition" refers to thepresence of dysplastic cells including carcinoma in situ, neoplastic,malignant, or tumor cells, or the like. For example, the specimen can becontacted or combined with a monoclonal antibody which recognizes adeterminant site on Le^(y) or Le^(x) such as a monoclonal antibody ofthe invention, e.g., as L15 or L17 antibody or an antibody havingsimilar characteristics such as, for example, those antibodies describedby Hakomori et al., supra, Fukushi et al., supra, Chia et al., supra,Abe et al., supra, Lloyd et al., supra, and Brown, et al., supra. Thecontact is carried out under conditions for binding of the antibody tothe malignant cells. After contact, the presence of binding of theantibody to the malignant cells in the specimen is observed. That is,the specimen is examined for immune complexes of the antibody and theantigenic site. This immune complex formation is related to the presenceof malignant cells in the specimen.

A particular example, by way of illustration and not limitation, of amethod in accordance with the invention is a method for the detection oftumor cells in excised tissue. The above method is applied to a specimenwhich is a section of the tumor obtained after removal of the tumor. Thetumor that is excised is treated to obtain sections, which treatmentinitially involves freezing the tumor or tissue, normally freezingimmediately after excision. The frozen layer of tissue is then cut intosections using, for example, a cryostat.

The section of the tumor obtained as described above is contacted with amonoclonal antibody of the invention and then with a second antibodydirected against the above monoclonal antibody, which second antibody islabeled with a detectable label.

The excised specimen, e.g., the section of the tumor, is contacted withthe first monoclonal antibody under conditions for binding of theantibody to the malignant cells. The incubation is generally conductedin an aqueous medium such as, for example, phosphate buffered salinecontaining a small amount of sodium azide, in a suitable container suchas, for example, a glass petri dish, for a period from about 15 to 30minutes at a temperature of from about 20° to 30° C. The amount ofantibody employed is usually sufficient to provide detectable binding,i.e., to provide a detectable number of immune complexes between theantibody and the determinant or antigenic site in question.

Following the incubation, the section is washed to reduce or eliminatenon-specifically bound antibody and then is examined to observe theabove-mentioned complexes which result from binding of the monoclonalantibody to the cells of the specimen possessing the antigenic site. Thebinding is related to the presence of malignant cells in the section.Accordingly, binding is determined, for example, by contacting thespecimen with a labeled specific binding partner for the monoclonalantibody. The label is capable of producing a detectable signal and maybe a radioactive label, a chromophore such as a fluorescer, an enzyme,or the like.

An example of a technique employing the above approach isimmunofluorescence staining. In this technique frozen sections of thetumor are fixed on a glass slide with acetone and are incubated with themonoclonal antibody in, for example, a petri dish. After washing with anappropriate buffer such as, for example, phosphate-buffered saline, thesection is placed on a petri dish and contacted with the labeledspecific binding partner for the monoclonal antibody, which may be, forexample, a labeled antibody specific for the monoclonal antibodyemployed. Since, for the most part, the monoclonal antibody will bederived from a murine source, a labeled anti-mouse immunoglobulinspecific for the monoclonal antibody may be employed. Suchimmunoglobulins may be raised according to standard techniques byinjecting a suitable host with murine antibody, waiting for anappropriate time, and harvesting the anti-mouse immunoglobulins from theblood of the injected host.

After a second washing of the slide with, for example, an aqueousbuffer, the sections may be covered with a fluorescent antibody mountingfluid and a coverslip and then examined with a fluorescence microscopeto determine the binding of the monoclonal antibody to the section. Thedetermination of the binding also may include an identification of thelocation of such binding within the specimen.

The binding of the monoclonal antibody to the specimen may also bedetermined by employing a monoclonal antibody which is covalentlyconjugated to a label capable of producing a detectable signal, such asa radioactive entity, a chromophore including dyes and fluorescers, oran enzyme. The number of labels employed per antibody is generallydetermined by the requirements of the diagnostic method in which thelabeled antibody is employed and the availability of sites for linkingthe label to the antibody.

Methods for conjugating labels to antibodies and antibody fragments arewell-known in the art. Such methods may be found in U.S. Pat. Nos.4,220,450; 4,235,869; 3,935,074; and 3,996,345.

Another example of a technique in which the monoclonal antibodies of theinvention can be employed is the detection of molecules bearing theLe^(x) and Le^(y) determinants in body fluids including plasma andserum. For this purpose, the antibody of the invention may be used aloneor in conjunction with another antibody directed against a separatedeterminant.

Still another example of a technique in which the monoclonal antibody ofthe invention can be employed is immunoperoxidase labeling (Sternberger,Immunocytochemistry, John Wiley & Sons, New York, 1979, pp: 104-169 asmodified by Garrigues et al., Int. J. Cancer (1982) 29: 511-515). Thetissue to be tested is fixed with a suitable solvent, such as acetone,on a support, such as a glass slide. Next, the tissue is incubated withthe monoclonal antibody and then washed free of unbound antibody. Then,the tissue is incubated with rabbit anti-mouse IgG, washed to removeunbound antibody, combined with mouse peroxidase-anti-peroxidasecomplex, washed to remove unbound conjugate, and then treated withsubstrate for the enzyme. Following this treatment the slide is examinedfor a detectable signal.

The antibodies of the invention may be used in a method of determiningthe presence of a malignant condition, for instance in an exfoliativecell specimen from the lung, such as sputum or in a cervical smear. Bythe term "exfoliative" is meant that the specimen comprises isolatedcells or clumps of cells obtained by scraping or washing the surface oftissue, which cells are removed individually or in scales or laminae.The exfoliative cell specimen is to be distinguished from excised tissuesuch as that obtained by biopsy. Contact between the specimen and theantibody is made under conditions for binding of the antibody to theantigenic site. After contact, the presence or absence of binding of theantibody to the antigenic site is determined and is related to thepresence of a malignant condition in the lung.

To determine the presence of a malignancy in the lung, a sputum samplewould provide the exfoliative cell specimen to be used in the method.The method may find utility in the detection of a malignant condition inexfoliative cell specimens from the bronchus, gastro-intestinal tractincluding oral pharynx, mouth, etc.

The exfoliative cell specimen is next contacted with the aforementionedantibodies under conditions for binding of the antibody to the specificantigenic site in the specimen to form antigen-antibody complexes. Thisantigenic site may be present on cells or cell fragments in thespecimen. Generally, the specimen is placed on an appropriate support,such as, for example, a slide, usually glass, or some other suitablematerial. The exfoliative cell specimen is generally smeared on theslide to provide a thin layer of the specimen on the surface of theslide. The contact between the antibody and the specimen is generallycarried out in an aqueous buffered medium. The buffers which may beemployed include phosphate, tris, bicarbonate, etc. The pH is related tothe nature of the specimen and the antibody, and is generally in therange of from about 5 to 8. The aqueous medium may additionally containorganic polar solvents in an amount of from about 0 to 40%. The organicpolar solvents are water soluble and generally have from about 1 to 10carbon atoms and from about 1 to 4 oxygen atoms. The antibody will bepresent in the aqueous medium at a concentration of about 1 to 100μg/ml, preferably from about 10 to 20 μg/ml. The temperature during thecontact of the specimen with the antibody is usually from about 4° to40° C., preferably about 10° to 30° C. The period of contact is usuallyfrom about 15 to 120 minutes, preferably from about 30 to 60 minutes.

After the period of contact between the specimen and the antibody, thesupport is generally treated to remove unreacted antibody. Normally,this is accomplished by washing the support with an aqueous, usuallybuffered, medium. In general, the amount of wash solution should besufficient to remove the unreacted antibody.

Next, the presence of antibody bound to the antigenic site in thespecimen, which binding is related to the presence of a malignantcondition at the locus, is observed. That is, the specimen is examinedto determine the number of antigen-antibody (immune) complexes formed.It should be noted that in some instances very small numbers of theantigenic site in question may be found in the exfoliative cellspecimen. However, in a malignant condition, large numbers of theantigenic site will be present and this latter condition is readilydistinguishable by this method over a non-malignant condition because alarge number of antigen-antibody complexes will be measurable where amalignant condition exists. To make the determination of the presence ofbinding, means for producing a detectable signal is incorporated intothe assay system. For example, one may conjugate the antibody employedin the assay to a label which is capable of producing a detectablesignal. The label may be a radioactive entity, a chromophore includingdyes and fluorescers, an enzyme, or the like. The number of labelsemployed for the antibody is generally determined by the requirements ofthe method and the availability of sites for linking the label to theantibody.

Alternatively, one may contact the washed slide with a labeled specificbinding partner for the antibody, which may be, for example, a labeledantibody specific for the antibody employed. Where the monoclonalantibody is derived from a murine source, a labeled anti-mouseimmunoglobulin specific for the antibody employed in the method may beused. Such immunoglobulins may be raised according to standardtechniques by injecting a suitable host with the monoclonal antibody,waiting for an appropriate time, and harvesting the anti-mouseimmunoglobulins from the blood of the injected host. When a labeledspecific binding partner for the antibody is employed, the slide must bewashed again with an aqueous medium prior to examining the slide forfluorescence.

To determine the presence of binding between the antibody and the cellspecimen where a fluorescer label is used, one may examine the slide forfluorescence, usually employing a fluorescence microscope. Where a labelother than a fluorescer is employed, one may examine the slide or thespecimen for the formation of a precipitate, a color, or the like.

The above description is directed primarily to the use of the antibodiesof the invention in immunofluorescence techniques. However, theantibodies of the invention can be used in most assays involvingantigen-antibody reactions. The assays may be homogeneous orheterogeneous. In a homogeneous assay approach, the specimen may bebiological fluid such as serum, urine, and the like or the specimen maybe lysed and clarified to remove debris. The immunological reactionusually involves the specific antibody, a labeled analyte, and thesample of interest. The signal arising from the label is modified,directly or indirectly, upon the binding of the antibody to the labeledanalyte. Both the immunological reaction and detection of the extentthereof are carried out in a homogeneous solution. Immunochemical labelswhich may be employed include free radicals, fluorescent dyes, enzymes,bacteriophages, coenzymes, and so forth.

In a heterogeneous assay approach, the reagents are usually thespecimen, the specific antibody, and means for producing a detectablesignal. The specimen is generally placed on a support, such as a plateor a slide, and contacted with the antibody in a liquid phase. Thesupport is then separated from the liquid phase and either the supportphase or the liquid phase is examined for a detectable signal employingmeans for producing such signal. The signal is related to the presenceof the analyte in the specimen. Means for producing a detectable signalincludes the use of radioactive labels, fluorescers, enzymes, and soforth. Exemplary of heterogeneous immunoassays are the radioimmunoassay,immunofluorescence methods, enzyme-linked immunoassays, and the like.

For a more detailed discussion of the above immunoassay techniques, see"Enzyme-Immunoassay," by Edward T. Maggio, CRC Press, Inc., Boca Raton,Fla., 1980. See also, for example, U.S. Pat. Nos. 3,690,834; 3,791,932;3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654; 3,935,074;3,984,533; 3,996,345; and 4,098,876, which listing is not intended to beexhaustive.

The antibodies of the invention can also be employed to image metastaticdeposits in human patients with NSCLC in a manner analogous to thatdescribed for malignant melanoma in J. Nucl. Med. (1983) 24: 123-129 andin J. Clin. Invest. (1983) 72: 2101-2114. The antibody or fragmentsthereof are radiolabelled and administered intravenously to a patientwho subsequently is imaged using, for example, a gamma camera or thelike.

While the above specific examples of methods in accordance with thepresent invention employ antibodies binding to specific determinantsites on the respective antigens and being of the IgM sub-classes from amurine source, this is not meant to be a limitation. The aboveantibodies and those antibodies having functional equivalency with theabove antibodies, whether from a murine source, other mammalian sourceincluding human, or other sources, or combinations thereof can beemployed in the method of this invention, as well as other isotypes. Bythe term "functional equivalency" is meant that the antibody is capableof binding to either of the respective above-described determinant sitesand capable of competing with a particular antibody of the invention forsuch site. That is, such antibody, when combined with a specimencontaining a cell or cell fragment or secreted antigen having suchdeterminant site, will bind to such determinant site and will block anantibody of the invention from binding to such site. Furthermore, sincethe Le^(y) and Le^(x) antigens can have more than one determinant site,the method of the invention includes the use of monoclonal antibodieswhich define determinant sites other than determinant sites defined bythe aforementioned monoclonal antibodies.

The invention also includes diagnostic kits for carrying out the methodsdisclosed above. In one embodiment, the diagnostic kit comprises (a) amonoclonal antibody more specifically defined above and (b) a conjugateof a specific binding partner for the above monoclonal antibody and alabel capable of producing a detectable signal. The reagents may alsoinclude ancillary agents such as buffering agents and proteinstabilizing agents, e.g., polysaccharides and the like. The diagnostickit may further include, where necessary, other members of thesignal-producing system of which system the label is a member, agentsfor reducing background interference in a test, control reagents,apparatus for conducting a test, and the like. In another embodiment,the diagnostic kit comprises a conjugate of a monoclonal antibody of theinvention and a label capable of producing a detectable signal.Ancillary agents as mentioned above may also be present.

The antibodies of the invention may be used therapeutically. Antibodieswith the proper biological properties can be useful directly astherapeutic agents Sears, et al, Contrl. Oncol. Karger, Basel, (1984)19: 180-192). Furthermore, antibodies can be bound to a toxin to form animmunotoxin or to a radioactive material or drug to form aradiopharmaceutical or pharmaceutical. Methods for producingimmunotoxins and radiopharmaceuticals of antibodies are well-known (see,for example, Cancer Treatment Reports (1984) 68: 317-328).

Another therapeutic use of the monoclonal antibody of the presentinvention is the immunization of a patient with an anti-idiotypicantibody raised by using one of the present monoclonal antibodies as animmunogen. Such immunization can induce an active anti-tumor activity(see, for example, Nepom et al.; Proc. Natl. Acad. Sci. U.S.A. (1984)81: 2864-2867. In a similar approach, the patient can be immunized withthe respective antigen in purified form or a modified form of therespective antigens.

An attractive aspect of the present invention is that the presentantibodies can be combined with other antibodies to NSCLC such as thosedisclosed in U.S. patent application Ser. Nos. 667,521 and 684,759,filed Nov. 2, 1984 and Dec. 21, 1984, respectively. The combination iseffective in detecting at least the four types of lung carcinomasmentioned above, namely, large cell undifferentiated lung carcinoma,small cell lung carcinoma, adenocarcinoma, and epidermoid carcinoma.

The monoclonal antibodies of the invention also define determinant siteson antigens associated with other carcinomas such as breast carcinomas.Consequently, the present antibodies can find use in diagnostic andtherapeutic products directed to such carcinomas.

EXAMPLES

The invention is further demonstrated by the following illustrativeExamples. A number of procedures employed will be described first.

Immunohistological Technique

For immunohistological studies on frozen sections, the unlabelledantibody technique of Sternberger in Immunochemistry, John Wiley & Sons,New York, 1979, pp: 104-169, as modified by Garrigues et al. in Int. J.Cancer (1982) 29: 511-515, was used. The target tissues for these testswere obtained at surgery and frozen within 4 hr of removal in isopentanewhich had been precooled in liquid nitrogen. Tissues were then stored inliquid nitrogen or at -70° C. until use. Rabbit anti-mouse IgG(Sternberger-Meyer Immunochemicals, Inc., Jarettsville, MD) was used ata dilution of 1/50. Mouse peroxidase-antiperoxidase complex (PAP,Sternberger-Meyer Immunochemicals, Inc.) containing 2 mg/ml ofspecifically purified PAP was used at a dilution of 1/80. Frozensections were prepared, dried, treated with acetone and dried (Garrigueset al., 1982). Sections to be used for histologic evaluation werestained with hematoxylin. To decrease nonspecific background, sectionswere preincubated with normal human serum diluted 1/5 (Garrigues et al.,1982). Mouse antibodies, goat anti-mouse IgG, and mouse PAP were dilutedin a solution of 10% normal human serum and 3% rabbit serum.

The staining procedure consisted of treating serial sections with eitherspecific or control antibody for 2.5 hr, incubating for 30 min withrabbit anti-mouse IgG diluted 1/50, and exposing for 30 min to mouse PAPcomplex diluted 1/80. After each treatment with antibody, the slideswere washed twice in phosphate buffered saline (PBS). Theimmunohistochemical reaction was developed with freshly prepared 0.05%3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) and0.01% hydrogen peroxide in 0.05M Tris buffer, pH 7.6 for 8 min. Furtherexposure to a 1% OsO₄ solution in distilled water for 20 min intensifiedthe stain. The sections were rinsed with water, dehydrated in alcohol,cleared in xylene, and mounted on slides.

The slides were each read under code and coded samples were checked byan independent investigator. Typical slides were photographed by usingdifferential interference contrast optics (Zeiss-Nomarski). The degreeof antibody staining was evaluated as 0 (no reactivity), + (few positivecells), ++ (at least one third of the cells positive), +++ (most cellspositive), ++++ (close to all cells strongly positive). Sincedifferences between + and 0 staining were less clear cut than between ++and + staining, it was decided to count as "positive" a staining gradedas ++ or greater. Both neoplastic and stroma cells were observed intumor samples; the staining recorded referred to that of the tumorcells, since the stroma cells were not stained at all, or were stainedmore weakly than the tumor cells.

Determination of Antigen Location

The subcellular localization of antigens was determined by measuringantibody binding to cells before or after permeabilization withnon-ionic detergent. Antibodies binding to the cell surface of intactcultured cells were identified by either direct binding assays with ¹²⁵I-labelled antibody (Brown et al., Proc. Natl. Acad. Sci. U.S.A. (1981)78: 539-543) or by indirect fluorescence using the (FACS) II cellsorter. Antibodies binding to intracellular locations were determined bydirect binding of ¹²⁵ I-labelled antibody to cells following fixationwith paraformaldehyde and subsequent permeabilization with the non-ionicdetergent NP-40.

Binding Assays

(a) For binding assays performed by using radiolabelled antibodies(Brown et al., supra), cultured cells (10⁶) were incubated at 4° C. for30 min with 10⁶ cpm of ¹²⁵ I-labelled antibody in 100 μl ofheat-activated (30 min at 56° C.) fetal calf serum in culture medium.After the addition of 5 ml of PBS, the cells were pelleted bycentrifugation for 10 min at 250×g. The supernatant was aspirated, andthe pellet was counted for ¹²⁵ I. To measure nonspecific binding,parallel incubations were performed with 10 μg of unlabelled antibody asa competitor (Brown, et al., supra). In some instances binding assayswere carried out in an analogous fashion on cells monolayers attached toplastic culture dishes.

(b) For binding assays performed on the FACS II cell sorter, cells wereremoved from their substrata using PBS containing 5 mM ethylenediaminetetraacetic acid (EDTA). Samples containing 1×10⁵ cells were incubatedfirst with monoclonal antibody at a concentration of 2 μg/ml followed byfluorescein-conjugated goat anti-mouse antibody at a 1:200 dilution.Cells were then washed and resuspended in culture medium. Immediatelyprior to FACS analysis, propidium iodide was added to a finalconcentration of 1 μg/ml to stain non-viable cells. During FACSanalysis, cells emitting red fluorescence were electronically gated outso that only viable cells were examined. The mean intensity offluorescein fluorescence was then determined for each antibody. Negativecontrols consisted of samples in which monoclonal antibody was omitted;positive controls consisted of monoclonal antibodies to HLA type 1histocompatibility antigens. Staining was regarded as positive if themean channel fluorescein was at least 3 times background.

Determination of Reactivity of Antibodies to Glycolipids

Antibodies were tested for reactivity to glycolipid antigens byincubation with purified glycolipids adsorbed to microtest wells (alongwith cholesterol and lecithin) and with thin layer chromatography plateson which glycolipids had been fractionated. Bound antibody was detectedby incubation with antiserum to mouse immunoglobulin and radioiodinatedprotein A.

Isotype Determination

(a) Ouchterlony immunodiffusion

An aliquot of supernatant of particular hybridoma cells was placed intothe center well of a 2% agar plate. Monospecific rabbit anti-mouse Igisotype antibodies (Meloy) were placed in the outer wells and the platewas incubated for 2 hr at room temperature and overnight at 4° C.

(b) Flexible polyvinylchloride 96 well plates (Costar) were coated with0.1 mg/ml goat anti-mouse Ig antibodies for 2 hr at 37° C. andcountercoated with a 3% BSA solution for 2 hr at 37° C. The hybridomasupernatant was then incubated at 37° C. for 2 h. After washing with PBSbovine serum albumin (BSA) plates were incubated at 37° C. for 2 hr withmonospecific rabbit anti-mouse Ig isotype antibodies coupled toperoxidase (Zymed). After washing, plates were incubated with 1 mg/mlorthophenylenediamine and 0.03% H₂ O₂ in 0.1M citrate buffer pH 4.5.Optical density at 630 nm was determined on a Dynatec ELISA platereader.

Staphylococcal Protein A Binding Assay

Microtiter wells were incubated with 5% FCS in PBS plus 0.02% NaN₃ andthe supernatant was aspirated. Twenty-five μl of a suspension of tumorcells (2×10⁷ cells/ml) were added to each well and incubated with 25 μlof a particular antibody for 1 hr at room temperature. The plates werecentrifuged at 1200 rpm for 7 min, washed twice with 50% NCS/PBS/NaN₃and 25 μl ¹²⁵ I-staphylococcal protein A (about 50,000 cpm/25 l) wereadded. The plates were incubated for 1 hr at 25° C., washed twice with5% NCS/PBS/NaN₃ and dried. The bottom of the wells were cut off andcounted in a gamma counter.

EXAMPLE

Preparation of Monoclonal Antibodies

Monoclonal antibodies were produced by immunizing 3-month-old BALB/cmice with cells from pleural effusions from patients with metastaticnon-small cell lung carcinoma. The immunizations were performed byinjecting the mice intraperitoneally 3-4 times with approximately 10⁷cells. Three days after the last immunization, the spleens were removed,suspended in culture medium and fused with NS1 mouse myeloma cells(Kohler and Milstein, supra). The mixtures were seeded to form lowdensity cultures originating from single fused cells (clones); thetechniques used for the hybridization have been previously described byYeh, et al., Int. J. Cancer (1979) 29: 269-275.

Supernatants from hybrid cells were screened by using both an ELISAassay and an autoradiographic indirect ¹²⁵ I-labelled protein A Assay(Brown et al., J. Immunol. Meth. (1979) 31: 201-209 against extractsfrom the tumors used for immunization which contained, i.a., cellmembranes. These extracts were prepared using a procedure modified fromColcher et al., Cancer Res., (1981) 42: 1451-1459; Yeh et al., supra.For this, tissues were washed with PBS and suspended, which for intacttumors was done by pressing through a stainless steel screen. After this1 mM NaHCO₃ containing 1 mM phenylmethylsulfonylfluoride(Calbiochem-Behring Corp., San Diego, CA) was added, and the materialwas then homogenized on ice, with 50 strokes of the B pestle of a Douncehomogenizer. After centrifugation for 15 min at 27,000×g, thesupernatant was removed, and the pellet was resuspended in PBS,sonicated for 1 min, and stored at -70° C.

Hybridomas which produced antibodies binding to the cell membraneextracts were cloned, expanded in vitro, and further tested for antibodyspecificity. This testing was carried out by using theImmunohistological Technique described above, in which the ability ofthe antibodies to bind to frozen sections of lung carcinomas, othertumors and normal human tissues were tested. Those hybridomas whichproduced antibody of apparent specificity for human lung cancer wererecloned, expanded and injected into pristane-primed 3-month old BALB/cmice, where they grew as ascites tumors.

Antibodies secreted into the ascites were purified on protein ASepharose (Ey et al., Immunochemistry (1978) 15: 429) or by gelfiltration in Sephacryl S-300. Purified antibodies were used for furthercharacterization which included additional specificity tests byimmunohistology, binding assays on intact cells to determine whichantibodies bound to the cell surface, and the radioimmunoprecipitationtests as described above.

Monoclonal antibodies L15 and L17 were produced from the correspondinghybridoma as described above. These antibodies exhibited the propertiesindicated above in this specification.

Antibodies L15 and L17 were subjected to immunohistological techniquedescribed above.

Antibody L15 bound strongly to NSCLC cells and did not bind to thefollowing cells: colon carcinoma, breast carcinoma, normal heart,leukocytes, brain, colon, kidney, spleen, skin, and liver.

Antibody L17 bound strongly to NSCLC cells, colon carcinoma cells, andbreast carcinoma cells. L17 exhibited weak binding with leukocytes andno binding with the following cells: normal heart, brain, colon, kidney,spleen, skin, liver.

The cell lines, designated L15 and L17, were both deposited at theA.T.C.C. on Mar. 1, 1985, and received accession numbers HB8738 andHB8739, respectively.

The invention has been described in detail with particular reference tothe above embodiments. It will be understood, however, that variationsand modifications can be effected within the spirit and scope of theinvention.

What is claimed is:
 1. A method for determining the presence of amalignant condition in the lung of a subject, which comprises examiningtissue from said subject, for the presence of a carbohydrate antigenwhich is Le^(y) antigen or an antigen having the characteristics ofLe^(y) antigen.
 2. The method of claim 1 wherein said tissue is selectedfrom the group consisting of excised tissue, exfoliative tissue, andbody fluids.
 3. A method for determining the presence of a malignantcondition in the lung of a subject, which comprises(a) contacting aspecimen of tissue obtained from said subject with a monoclonal antibodywhich defines a determinant site on an antigen which is Le^(y) antigenor an antigen having the characteristics of Le^(y) for binding of saidantibody to said specimen and (b) observing the presence of binding ofsaid antibody to said specimen, said binding being related to thepresence of a malignant condition in the lung.
 4. The method of claim 3wherein the extent of binding is determined by contacting said specimenwith a conjugate of a label and specific binding partner for saidmonoclonal antibody, said label being capable of producing a detectablesignal.
 5. The method of claim 4 wherein the labeled specific bindingpartner for said monoclonal antibody is an antibody specific for saidmonoclonal antibody.
 6. The method of claim 5 wherein the label is achromophore.
 7. The method of claim 3 wherein the monoclonal antibody isconjugated to a label capable of producing a detectable signal.
 8. Themethod of claim 3 wherein said antibody is L15 antibody.
 9. The methodof claim 3 wherein said tissue is selected from the group consisting ofexcised tissue, exfoliative tissue, and body fluids.
 10. A monoclonalantibody designated L15 produced by a hybridoma cell line having thecharacteristics of HB 8738 as deposited with the ATCC, the antigencombining site of which defines Le^(y), a surface carbohydrate antigencharacteristic of human non-small cell lung carcinoma.
 11. An Fab,F(ab')₂ or Fv fragment of the monoclonal antibody of claim
 10. 12. Themonoclonal antibody fragment of claim 11 conjugated to a label capableof producing a detectable signal.
 13. The monoclonal antibody fragmentof claim 12 in which the label is a fluorescer, a radiolabel, achromophore or an enzyme.
 14. The monoclonal antibody of claim 10conjugated to a label capable of producing a detectable signal.
 15. Themonoclonal antibody of claim 14 in which the label is a fluorescer, aradiolabel, a chromophore or an enzyme.
 16. A cell line which producesmonoclonal antibody L15.
 17. The cell line of claim 16 deposited withthe ATCC and assigned accession number HB 8738.